Plasmid for mouse ROSA26 locus for use with plasmid pBigT
Lead Inventors: Franklin David Costantini, PhDProblem or Unmet Need:In biomedical research, a very common technique is to alter a mouse's genome to induce or repress expression of a gene of interest. Although this technology has been around for many years, the actual laboratory work required to produce such a mouse is time-consuming and prone to failure. This technology consists of a plasmid that has been optimally designed to insert any DNA sequence of choice into the mouse genome at the ROSA26 locus. This locus is important because it has a high endogenous expression level throughout development; thus, the inserted sequence will be expressed globally, or can be restricted in time and/or space by using a second 'driver' mouse line. The technology consists of the generic targeting vector, called pROSA26-PA, which can be used in conjunction with pBigT (see IR #2446) to easily target the ROSA locus. A scientist wishing to create a gene insertion first assembles the desired sequence, subclones it into the pBigT vector, and then moves a larger fragment from pBigT into the pROSA26-PA vector. After this step, the final targeting sequence is extracted and inserted into the mouse genome (e.g., by embryonic stem cell transformation). This technology makes targeting of the ROSA26 locus straightforward by incorporating several features:o Rare-cutting restriction enzyme sites AscI and PacI allow for 'sticky-end' ligation, obviating the need for more time-consuming types of ligation.o The companion pBigT plasmid includes useful ancillary sequences such as PGK-neoPGK-pA and loxP sites, for use with Cre recombinase. This technology was amply demonstrated in the creation of Cre reporter strains using gene-encoded fluorescent tags
The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize ?-gal in living tissue.
These targeting constructs are used to create mice suitable for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes using fluorescent protein marker expression.
The technology consists of the generic targeting vector, called pROSA26-PA, which can be used in conjunction with pBigT (see None
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